Myoglobin (Mb) is the monomeric heme-binding oxygen carrier protein of red muscle. We will examine the regulation of mb gene expression in various tissues. Recombinant DNA techniques will be used to select cDNA clones specific for mb for partially purified mRNA of embryonic chick thigh muscle. Labelled cloned cDNA will provide a probe with which to quantitate nuclear and cytoplasmic mb RNA levels in red and white muscle and in non-muscle tissues. The labelled cDNA will also be used to identify library clones containing the genomic mb sequences. Their structure will be characterized by restriction mapping, R-looping, Southern blotting, and partial DNA sequence analysis. Protein gel blotting and affinity chromatography on mb-DNA columns will be used to identify and purify proteins exhibiting specific binding of the mb region. We will attempt to identify proteins responsible for activation of mb genes by assaying for differential distribution of mb-specific DNA binding proteins between mb-producing and non-producing tissue, and by their ability to restore DNAse sensitivity to mb genes of reconstituted chromatin. The interaction between these proteins and their high-affinity binding sites will be examined. A cell culture system will be established to investigate the role of extracellular factors on regulation of the mb genes. These studies should yield important basic knowledge about eucaryotic regulatory processes in general and regulation of muscle proteins in particular, providing the baseline with which to compare alterations in these processes caused by myopathies.